Abstract
Proper differentiation of sperm from germline stem cells, essential for production of the next generation, requires dramatic changes in gene expression that drive remodeling of almost all cellular components, from chromatin to organelles to cell shape itself. Here we provide a single nucleus and single cell RNA-seq resource covering all of spermatogenesis in Drosophila starting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study (Li et al., 2022). With over 44,000 nuclei and 6,000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. We justify assignment of key germline and somatic cell types using combinations of known markers, in situ hybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, we provide datasets compatible with commonly used software such as Seurat and Monocle. The foundation provided here will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for function in vivo.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵# ’contributed equally’ justification: AAR, GSV, SRS, SM, JMF, JMV, SP, JRG, and MRG each took the lead on a particular aspect of the coding, analysis, documentation, construction of at least one specific Figure and or its supplement, and/or assembly of supporting source data, and also re-iteratively drafted sections of the narrative reporting that work.
First, a number of new markers were identified from the cluster analysis, and expression was verified using FISH or reporter lines. Second, independent clustering analyses verified the identity of the hub. Finally, new FISH analysis directly demonstrates the predicted shift from active transcription in spermatocytes to stored cytoplasmic mRNA in spermatids.